Two long-lasting human monoclonal antibodies cross-react with monkeypox virus A35 antigen

Supplementary information Materials and Methods Study approval and biological samples This study was approved by the Ethics Committee of Shenzhen Third People’s Hospital, China (approval number: 2021-030). The participants had provided written informed consent for sample collection and subsequent analysis. All plasma and peripheral blood mononuclear cells (PBMCs) samples were stored at the BioBank of Shenzhen Third People’s Hospital. All plasma samples were stored at -80 °C and heat-inactivated at 56 °C for 1 h before use. PBMCs were maintained in freezing medium and stored in liquid nitrogen before use.

maintained in freezing medium and stored in liquid nitrogen before use.

Enzyme linked immunosorbent assay (ELISA)
The native recombinant proteins of MPXV, VACV, Epstein-Barr virus, and Influenza B virus (Sino Biological and AntibodySystem) or denatured by treatment with the denaturing buffer (New England Biolabs) at 95 °C for 10 mins were coated into 96-well plates at 4 °C overnight. The plates were washed with PBST buffer and blocked with 5% skim milk and 2% bovine albumin in PBS at RT for 1 h. Diluted plasma samples and monoclonal antibodies (mAbs) were added into wells and incubated at 37 °C for 1 h. The plates were washed and HRP conjugated goat anti human IgG antibodies (ZSGB-BIO) were added and then incubated at 37 °C for 30 mins (plasma) and for 1 h (mAbs). Finally, the TMB substrate (Sangon Biotech) was added and incubated at RT for 5 mins (plasma) and for 20 mins (mAbs) and the reaction was stopped by 2M H 2 SO 4 .
The readout was detected at a wave length of 450 nm. The 50% effective concentration (EC 50 ) values of tested mAbs were calculated using GraphPad Prism 8 software by log (agonist) vs. response --Variable slope (four parameters) model.

Competition ELISA
The 96-well plates were coated with MPXV A35 protein (Sino Biological) at 4 °C overnight. The plates were washed with PBST buffer and blocked with 5% skim milk and 2% bovine albumin in PBS at RT for 1 h. Serially diluted mAbs were mixed with HRP (Abcam) conjugated MPXV-mAb 975 or MPXV-mAb 981 in 2 equal volume and then added to the plates and incubated at 37 °C for 1 h.
Finally, the TMB substrate (Sangon Biotech) was added and incubated at RT for 20 mins and the reaction was stopped by 2M H 2 SO 4 . The readout was detected at a wave length of 450 nm. The competitive effect was determined by comparing the ratio of different mAbs to the negative control mAb (VRC01, an HIV-1 mAb).

Neutralization assay
For live VACV preparations. Vero E6 cells in T75 were infected at a multiplicity of infection (MOI) of 0.1. Cells were harvested at Day 2, and virus was isolated by rapidly freeze-thawing the cell pellet three times in a volume of 5 mL of DMEM supplemented with 2% heat-inactivated FBS (D-2). Cell debris was removed by centrifugation at 800 g for 5 mins. The virus containing supernatant was aliquoted and stored at -80 °C.
To test the neutralization activity of the heat inactivated human plasma, Vero E6 cells were seeded at 1.5 × 10 4 cells/well into 96-well plates and used the following day. Plasma samples 10-fold diluted in D-2 medium with 2% sterile guinea pig complement (Beijing Bersee Science and Technology Co.,Ltd) were mixed with equal volume of diluted live VACV and then incubated at 37 °C for 1 h. Medium from 96-well plates was aspirated, and plasma-virus mixture was added and allowed to adsorb at 37 °C for 1 h. Cells were rinsed with warm PBS, overlaid with D-2 medium, and the plates were incubated at 37 °C for 8 h. Then cells were fixed with 4% paraformaldehyde solution, permeabilized with Perm/Wash buffer (BD Biosciences) containing 0.1% Triton X-100, incubated with the HRP-conjugated anti-VACV polyclonal antibodies (Invitrogen) at 4 °C overnight. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences). The numbers of VACV foci were calculated using an EliSpot reader (Cellular Technology Ltd).

Isolation of mAbs from MPXV-donor 3 and MPXV-donor 42
Thawed PBMCs were stained with an antibody cocktail including CD19-PE-Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue, CD27-APC-H7, and IgG-FITC (BD Biosciences) to gate IgG+ memory B cells. MPXV-A35 with a C-3 His tag (Sino Biological) was used as a probe to sort antigen-specific single cells. To exclude the nonspecific staining, two anti-His secondary antibodies labeled with APC and PE (Abcam) were both used to recognize the MPXV-A35 bait. The flow cytometric data were acquired on BD FACSymphony S6 (BD Biosciences) and analyzed using FlowJo software (TreeStar). Single B cells were sorted into 96-well PCR plates containing lysis buffer followed by RT-PCR and nested PCR to amplify variable regions of heavy-and light-chain. Variable genes were sequenced by Sangon Biotech, synthesized by GenScript, and then separately cloned into full-length IgG1 heavy and light chain expression vectors. Monoclonal antibodies were expressed by co-transfection of 293 F cells with paired heavy-and light-chain plasmids and purified from the culture supernatants using protein A column (GenScript).

Binding affinity analysis by surface plasmon resonance (SPR)
The binding assays of mAbs to the MPXV-A35 protein (Sino Biological) were performed using the Biacore 8K system (GE Healthcare). Specifically, one flow cell of the CM5 sensor chips were covalently coated with MPXV-A35 in 10 mM sodium acetate buffer (pH 5.0) for a final RU (response units) around 250, whereas the other flow cell was left uncoated and blocked as a control. All the assays were run at a flow rate of 30 µL/min in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20). Serially diluted antibodies were injected for 60 s respectively and the resulting data were fit in a 1:1 binding model with Biacore Evaluation software (GE Healthcare). Every measurement was performed two times and the individual values were used to produce the mean affinity constant.

Epitope analysis by flow cytometry
The 293